Project Summary ?Microtubule bundling: effects of detyrosination and changes in cardiomyocyte mechanics? The cardiac microtubule cytoskeleton functions include, among other things, modulating myocyte structural and mechanical properties. In certain forms of heart disease extensive proliferation and post-translational modification of the microtubule network occurs. The post-translational modification of the network, particularly detyrosination, is strikingly correlated with diminished cardiac output. Our recent work has shown that the upregulation of detyrosination alone is sufficient to impair contraction in unloaded, isolated myocytes by increasing internal mechanical resistance due to the formation of energetically unfavorable buckles. The availability of pharmaceutical agents targeting detyrosination without perturbing the overall microtubule cytoskeleton make it an attractive therapeutic target in heart disease. As detail of the underlying mechanism has focused on detyrosination, the possibility of MAP-mediated bundling became apparent. Several MAPs interact preferentially with detyrosinated tubulin and can mediate the formation of microtubule bundles. The formation of microtubule bundles would be expected to significantly increase the stiffness of the resulting structure, and would increase the potential for microtubules to act as mechanical resistors. The applicant and mentor made the first observation of microtubule buckles in beating adult cardiomyocytes in work accepted for publication this spring (Robison et al. Science 2016). This work included several major advances. First, we developed genetically encoded tools for labeling and manipulating detyrosination in live myocytes. These tools permit highly specific manipulation of levels of detyrosination which will allow us to characterize the effects of detyrosination on bundling by electron microscopy. Second, we used these tools in conjunction with newly commercialized point spread function reconstruction techniques, allowing us to image microtubule behavior on the timescale of a heartbeat. Further advances in PSF reconstruction scheduled to be installed on our microscope will be available for this work and allow us to simultaneously record the sarcomere spacing proximal to the buckling microtubule. Third, we have developed a mathematical model relating microtubule buckling to sarcomere shortening. This model will be refined by the characterization of microtubule bundles in various states of the microtubule network and make testable predictions about the effects of bundling on contractility. In this proposal we will test the hypothesis that detyrosination increases microtubule bundling and that this bundling contributes to mechanical resistance. We have 2 major goals of our proposal: 1) to determine if and how increasing detyrosination alters the bundling state of the cardiac microtubule cytoskeleton; 2) to determine if changes in bundling alone are able to impart meaningful mechanical resistance to the myocyte. Our team of cardiomyocyte physiologists, cytoskeletal biologists, and a cardiac physician scientist are ideally suited to achieving these goals. Completed successfully, this work will provide mechanistic insight into how the structure of the microtubule cytoskeleton is influenced by posttranslational modifications and how that structure affects the function of the myocyte as a whole.